JP4694760B2 - microscope - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a microscope using laser light or the like, and more particularly to a microscope having both functions of a confocal laser scanning microscope and a total reflection microscope.
[0002]
[Prior art]
Conventional microscopes using laser light include confocal laser scanning microscopes and total reflection microscopes. All of them are used to observe and detect fluorescence generated mainly in a fluorescent specimen by irradiating a biological specimen or the like with a laser beam to excite it.
[0003]
A confocal laser scanning microscope irradiates a sample with a laser beam spot focused near the diffraction limit of the objective lens, and irradiates the sample with a microscopic aperture at the optical conjugate position. Detect through.
[0004]
In addition, the total reflection microscope makes the illumination light incident on the specimen that is in close contact with the cover glass from the cover glass side, and the illumination light is incident on the boundary surface between the cover glass and the specimen at a critical angle above the critical angle at which the incident light is totally reflected. The evanescent wave generated in the region is used to excite the vicinity of the boundary surface of the sample to generate fluorescence, and the fluorescence is detected using a CCD camera or the like.
[0005]
The above-mentioned two types of microscopes use laser light as an illumination light source because it is necessary to focus illumination light on a specimen or an entrance pupil surface of an objective lens, but they are used only as independent microscopes. Yes.
[0006]
[Problems to be solved by the invention]
In recent years, there has been a demand for a microscope that can be used as a confocal laser scanning microscope and a total reflection microscope with a single microscope. Until now, in order to meet these requirements, confocal laser scanning microscopes and total reflection microscopes have been purchased separately and their optical systems have been combined, so almost two microscopes will be purchased. There is a problem that the apparatus becomes large and expensive at the same time.
[0007]
Further, when switching between the confocal laser scanning microscope optical system and the total reflection microscope optical system, there is a problem that the operation is complicated and the mechanism for switching becomes complicated.
[0008]
The present invention has been made in view of the above problems, and provides a microscope that can be used for both a confocal laser scanning microscope and a total reflection microscope by using simple switching of an optical member, and can also share a control device and the like. The purpose is that.
[0009]
[Means for Solving the Problems]
In order to achieve the above object, in the present invention, a laser illumination light source,
An objective lens;
A scanner disposed between the laser illumination light source and the objective lens, and scanning the illumination light from the laser illumination light source in at least one direction;
A first relay lens system fixedly disposed between the scanner and the objective lens;
A second relay lens system disposed between the objective lens and the first relay lens system;
A third relay lens system arranged to be interchangeable with the second relay lens system;
The focal length of the second relay lens system is larger than the focal length of the first relay lens system,
When prior Symbol second relay lens system is inserted in the optical axis, the second relay lens system, the illumination light from the first relay lens system and the second relay lens system the objective lens Through the first relay lens system and the second relay lens system, the light deflection unit of the scanner and the entrance pupil plane of the objective lens. Is substantially optically conjugated, and the illumination light can scan and illuminate the specimen by the scanner,
When the third relay lens system is inserted into the optical axis, the third relay lens system transmits the illumination light from the first relay lens system to the objective lens via the third relay lens. The first relay lens system and the third relay lens system are arranged so as to collect light near the entrance pupil plane, and the optical deflection unit of the scanner and the focal plane on the sample side of the objective lens are approximately optically conjugate. The microscope is characterized in that the illumination light can totally illuminate the specimen.
[0011]
In the present invention, a laser illumination light source, an objective lens, a scanner that is disposed between the laser illumination light source and the objective lens, and scans illumination light from the laser illumination light source in at least one direction;
A first relay lens system disposed between the scanner and the objective lens;
A fixed relay lens system for condensing the illumination light from the first relay lens system on a focal plane of the objective lens on the sample side of the objective lens ;
A third relay lens system for condensing the illumination light from the first relay lens system in the vicinity of an entrance pupil plane of the objective lens;
The third relay lens system is detachable between the first relay lens system and the fixed relay lens system,
When the third relay lens system is removed from the optical axis , the optical deflection unit of the scanner and the entrance pupil plane of the objective lens are substantially optically controlled by the first relay lens system and the fixed relay lens system. The illumination light can scan and illuminate the specimen by the scanner,
When the third relay lens system is inserted into the optical axis, the first relay lens system, the fixed relay lens system, and the third relay lens system allow the light deflection unit of the scanner and the objective lens to A microscope is characterized in that the focal plane on the specimen side is substantially optically conjugate, and the illumination light can totally illuminate the specimen.
[0013]
In the microscope of the present invention, a first observation optical system provided between the laser illumination light source and the scanner,
An observation optical path switching optical element detachably provided between the objective lens and the third relay lens system;
A second observation optical system provided in the vicinity of the observation optical path switching optical element,
When the second relay lens system or the fixed relay lens system is inserted in the optical axis and the observation optical path switching optical element is removed from the optical axis, light from the specimen is reflected in the first observation optical system. Can be observed
When the third relay lens system is inserted into the optical axis and the observation optical path switching optical element is inserted into the optical axis, light from the specimen passes through the observation optical path switching optical element. It is desirable to be able to observe with this observation optical system.
[0014]
In the microscope of the present invention, it is desirable that the observation optical path switching optical element is composed of a beam splitter and an illumination light cut filter arranged at approximately 45 degrees with respect to the optical axis.
[0015]
In the microscope of the present invention, it is preferable that the third relay lens system is movable in the optical axis direction in order to focus the illumination light near the entrance pupil plane of the objective lens.
[0016]
In the microscope of the present invention, only when the third relay lens system is inserted into the optical axis, the insertion / removal of the third relay lens system is performed such that the observation optical path switching optical element is inserted into the optical axis. It is desirable that the operation and the insertion / removal operation of the observation optical path switching element are interlocked.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described with reference to the drawings.
[0018]
FIG. 1 shows a schematic configuration diagram when the microscope according to the first embodiment of the present invention is used as a confocal laser scanning microscope, and FIG. 2 shows the total reflection of the microscope according to the first embodiment of the present invention. The schematic block diagram at the time of using as a microscope is shown. FIG. 3 shows a schematic configuration diagram when the microscope according to the second embodiment of the present invention is used as a confocal laser scanning microscope, and FIG. 4 shows the total reflection of the microscope according to the second embodiment of the present invention. The schematic block diagram at the time of using as a microscope is shown.
(First embodiment)
1 and 2 are schematic configuration diagrams of the microscope according to the first embodiment of the present invention. This microscope is configured by combining a confocal laser scanning microscope and a total reflection microscope by inserting / removing or replacing optical members. It can be used for both. Hereinafter, each case of the confocal laser scanning microscope and the total reflection microscope will be described.
[0019]
In the case of using as a confocal laser scanning microscope, in FIG. 1, the illumination light 2 emitted from the laser illumination light source 1 enters the two-dimensional scanner 3 and is emitted from the two-dimensional scanner 3, and the first relay lens system 4 The light enters the objective lens 6 through the second relay lens system 5 and is condensed on the sample 7.
[0020]
Light (reflected light or fluorescence) from the specimen 7 is collected by the objective lens 6, passes through the same optical path as the illumination light 2, and is approximately 45 with respect to the optical axis between the laser illumination light source 1 and the two-dimensional scanner 3. The light beam is reflected by the beam splitter 8 arranged at a degree and imaged on the photomultiplier 11 via the imaging lens 9 and the pinhole 10 and observed with a monitor or the like (not shown). Here, the beam splitter 8, the imaging lens 9, the pinhole 10 and the photomultiplier 11 constitute a first observation optical system.
[0021]
The two-dimensional scanner 3 controls two scanners whose light deflection directions are orthogonal by the control circuit 12, and scans the illumination light 2 in the two-dimensional plane of the sample 7.
[0022]
The scanning range and scanning speed of the illumination light 2 are determined based on a control signal sent from the personal computer 13 to the control circuit 12.
[0023]
The first relay lens system 4 and the second relay lens system 5 are arranged so that the light deflection unit of the two-dimensional scanner 3 and the entrance pupil plane I of the objective lens 6 are substantially optically conjugate. Note that the light polarization portion of the two-dimensional scanner 3 may be the mirror surface when the two-dimensional scanner 3 is formed of a mirror.
[0024]
In this way, the microscope of the present invention can be used as a confocal laser scanning microscope.
[0025]
In the case of using as a total reflection microscope, in FIG. 2, the illumination light 2 emitted from the laser illumination light source 1 enters the two-dimensional scanner 3, is emitted from the two-dimensional scanner 3, and enters the first relay lens system 4. The light is condensed in the vicinity of the entrance pupil plane I of the objective lens 6 through the relay lens system 14 and the relay lens system 15 which are the third relay lens system, and is emitted from the objective lens 6. In the vicinity of the entrance pupil plane I, the pupil position is also included. Since the illumination light 2 is condensed in the vicinity of the entrance pupil plane I of the objective lens 6, the specimen 7 is irradiated with substantially parallel light from the objective lens 6.
[0026]
The illumination light 2 emitted from the objective lens 6 passes through the cover glass 16 and is irradiated on the sample 7. The specimen 7 is, for example, fluorescently stained cultured cells adsorbed on the cover glass 16 and filled with a salt solution for keeping the cells alive, fluorescently stained fixed cells, or fluorescently stained tissue sections. Things are used. The illumination range is, for example, about 100 μm in diameter when a 100 × objective lens 6 is used.
[0027]
The position at which the illumination light 2 is incident on the entrance pupil plane I of the objective lens 6 can be freely changed by controlling the scanning operation of the two-dimensional scanner 3 based on a control signal sent from the personal computer 13 to the control circuit 12. Can do.
[0028]
When the illumination light 2 is incident near the center of the entrance pupil plane I of the objective lens 6, the illumination light 2 is emitted from the vicinity of the center of the objective lens 6 in a substantially vertical direction. When the incident position of the illumination light 2 is moved from the center of the entrance pupil plane I of the objective lens 6 toward the peripheral portion by scanning control of the two-dimensional scanner 3, the emitted light from the objective lens 6 is inclined accordingly, and finally the objective The illumination light 2 emitted from the lens 6 is in a total reflection state in which it is totally reflected by the boundary surface S between the specimen 7 and the cover glass 16.
[0029]
In this total reflection state, an evanescent wave is generated in the vicinity of the boundary surface S between the sample 7 and the cover glass 11, and only the vicinity of the boundary surface S of the sample 7 is selectively excited by the evanescent wave.
[0030]
Further, while being in the total reflection state, the vicinity of the center of the visual field is illuminated with the illumination light 2, and this illumination position (area where the illumination light 2 is totally reflected) is the illumination light 2 on the entrance pupil plane I. Even if the incident position changes, it hardly changes.
[0031]
The light (fluorescence) from the specimen 7 excited by the evanescent wave is collected by the objective lens 6 and reflected by the beam splitter 17 arranged at about 45 degrees with respect to the optical axis, and the illumination light cut filter 18 and the image are formed. The image is formed on an image pickup device (for example, a CCD camera) 20 through the lens 19, processed by the image generation circuit 21, and displayed on the monitor of the personal computer 13. Here, the beam splitter 17 and the illumination light cut filter 18 constitute an optical path switching optical element 23b, and the imaging lens 19 and the imaging device 20 constitute a second observation optical system.
[0032]
In this way, the microscope of the present invention can be used as a total reflection microscope.
[0033]
Note that the first relay lens system 4 and the third relay lens system (14, 15) are arranged so that the light deflection section of the two-dimensional scanner 3 and the focal position on the sample side of the objective lens 6 are approximately optically conjugate. Has been. Note that the light polarization portion of the two-dimensional scanner 3 may be the mirror surface when the two-dimensional scanner 3 is formed of a mirror.
[0034]
In the first embodiment, in order to switch between the confocal laser scanning microscope and the total reflection microscope, the second relay lens system 5 and the third relay lens system (14, 15) are exchanged with respect to the optical axis. It is supported by a lens support member 22 that can be made. In the first embodiment, the lens support member 22 has a rotation mechanism, and the second relay lens system 5 and the third relay lens system (14, 15) are rotatably arranged around the rotation shaft 22a. (For example, a rotating turret). Then, by rotating the lens support member 22 about the rotation shaft 22a, the second relay lens system 5 and the third relay lens system (14, 15) can be exchanged on the optical axis.
[0035]
The lens support member 22 is formed to be movable in the optical axis direction after the third relay lens system (14, 15) is inserted into the optical axis. As a result, even when the position of the entrance pupil plane I of the objective lens 6 is shifted in the optical axis direction (for example, when the objective lens 6 is replaced), the illumination light 2 is near the entrance pupil plane I of the objective lens 6. It is possible to adjust so that the light is condensed.
[0036]
In addition, the optical path splitting optical element 23b that is integrally provided with the beam splitter 17 and the illumination light cut filter 18 that is used when observing the total reflection microscope is supported by an optical element support member 23 having a rotation mechanism. By inserting the optical path dividing optical element 23b into the optical axis by the optical element support member 23 and introducing light from the specimen into the second observation optical system, total reflection microscope observation becomes possible.
[0037]
As described above, the second relay lens system 5 and the third relay lens system (14, 15) can be exchanged, and the optical path switching optical element 23b can be inserted into and removed from the optical axis. Switching between total reflection microscope observations is possible.
[0038]
Note that a switching mechanism other than a rotation mechanism may be used for exchanging the second relay lens system 5 and the third relay lens system (14, 15) and inserting / removing the optical path switching optical element 234b.
[0039]
Further, the rotation mechanism and the like are respectively supported at predetermined locations of a microscope main body (not shown).
[0040]
The lens support member 22 and the optical element support member 23 may be configured so that their operations are interlocked using a gear mechanism or a motor drive.
[0041]
The interlocking operation is preferably configured so that the optical path switching optical element 23b is inserted into the optical axis when the third relay lens system (14, 15) is inserted into the optical axis.
(Second Embodiment)
Next, an optical scanning microscope according to a second embodiment of the present invention will be described with reference to the drawings.
[0042]
3 and 4 show a microscope according to the second embodiment of the present invention. The difference between the second embodiment and the first embodiment is that the second relay lens system 5 of the first embodiment is changed to a fixed relay lens system 25, and the insertion / removal position of the third relay lens system 24 is different. There is a difference. 3 and 4, the fixed relay lens system 25 corresponds to, for example, a lens such as a second objective lens of an infinite microscope that is commonly used in both the confocal laser scanning microscope and the total reflection microscope. In addition, the same code | symbol is attached | subjected to the member equivalent to 1st Embodiment and description is abbreviate | omitted.
[0043]
In the case of using as a confocal laser scanning microscope, in FIG. 3, the illumination light 2 emitted from the laser illumination light source 1 enters the two-dimensional scanner 3 and exits from the two-dimensional scanner 3, and the first relay lens system 4 The light enters the objective lens 6 through the fixed relay lens system 25 and is condensed on the sample 7.
[0044]
Light (reflected light or fluorescence) from the specimen 7 is collected by the objective lens 6, passes through the same optical path as the illumination light 2, and is approximately 45 with respect to the optical axis between the laser illumination light source 1 and the two-dimensional scanner 3. The light beam is reflected by the beam splitter 8 arranged at a degree and imaged on the photomultiplier 11 through the imaging lens 9 and the pinhole 10 and observed with a monitor or the like (not shown). Here, the beam splitter 8, the imaging lens 9, the pinhole 10 and the photomultiplier 11 constitute a first observation optical system.
[0045]
The control method of the two-dimensional scanner 3 is the same as that in the first embodiment, and a description thereof will be omitted.
[0046]
The first relay lens system 4 and the fixed relay lens system 25 are arranged so that the light deflection section of the two-dimensional scanner 3 and the entrance pupil plane I of the objective lens 6 are substantially optically conjugate. Note that the light polarization portion of the two-dimensional scanner 3 may be the mirror surface when the two-dimensional scanner 3 is formed of a mirror.
[0047]
In the case of using as a total reflection microscope, in FIG. 4, the illumination light 2 emitted from the laser illumination light source 1 enters the two-dimensional scanner 3 and exits from the two-dimensional scanner 3. The light is condensed in the vicinity of the entrance pupil plane I of the objective lens 6 through the 3-relay lens system 24 and the fixed relay lens system 25 and is emitted from the objective lens 6. In the vicinity of the entrance pupil plane I, the pupil position is also included.
[0048]
Since the illumination light 2 is collected near the entrance pupil plane I of the objective lens 6, substantially parallel light is emitted from the objective lens 6 toward the sample 7. The method for setting the total reflection illumination state is the same as in the first embodiment, and the description thereof is omitted.
[0049]
Then, the light (fluorescence) from the specimen 7 excited by the evanescent wave generated in the total reflection illumination state is collected by the objective lens 6 and reflected by the beam splitter 17 arranged at approximately 45 degrees with respect to the optical axis. The image is formed on an imaging device (for example, a CCD camera or the like) 20 through the illumination light cut filter 18 and the imaging lens 19, processed by the image generation circuit 21, and displayed on the monitor of the personal computer 13. Here, the beam splitter 17 and the illumination light cut filter 18 constitute an optical path switching optical element 23b, and the imaging lens 19 and the imaging device 20 constitute a second observation optical system.
[0050]
In this way, the microscope of the present invention can be used as a total reflection microscope.
[0051]
The first relay lens system 4, the third relay lens system 24, and the fixed relay lens system 25 are configured so that the focal position on the sample side of the light deflection unit of the two-dimensional scanner 3 and the objective lens 6 is approximately optically conjugate. Is arranged. Note that the light polarization portion of the two-dimensional scanner 3 may be the mirror surface when the two-dimensional scanner 3 is formed of a mirror.
[0052]
In the second embodiment, switching between the confocal laser scanning microscope and the total reflection microscope is performed by supporting the third relay lens system 24 on the lens support member 26, and switching between the first relay lens system 4 and the fixed relay lens system 25. This is done by inserting / removing the optical axis between them (moving laterally in the figure).
[0053]
The lens support member 26 is formed to be movable in the optical axis direction after the third relay lens system 24 is inserted into the optical axis. As a result, even when the position of the entrance pupil plane I of the objective lens 6 is shifted in the optical axis direction (for example, when the objective lens 6 is replaced), the illumination light 2 is near the entrance pupil plane I of the objective lens 6. It is possible to adjust so that the light is condensed.
[0054]
The third relay lens system 24 may be inserted / removed by a rotation mechanism or the like in addition to the above-described lateral movement.
[0055]
The optical path switching optical element 23b integrally provided with the beam splitter 17 and the illumination light cut filter 18 that is used when observing the total reflection microscope is supported by an optical element support member 23 having a rotation mechanism. By inserting the optical path dividing optical element 23b into the optical axis by the optical element support member 23 and introducing light from the specimen into the second observation optical system, total reflection microscope observation is possible.
[0056]
As described above, the third relay lens system 24 is detachably disposed between the first relay lens system 4 and the second relay lens system 5, and the optical path switching optical element 23b is detachably disposed on the optical axis. This makes it possible to switch between confocal laser microscope observation and total reflection microscope observation.
[0057]
Note that a switching mechanism other than the rotation mechanism may be used to insert and remove the optical element with respect to the optical axis.
[0058]
Further, the rotation mechanism and the like are respectively supported at predetermined locations of a microscope main body (not shown).
[0059]
The lens support member 26 and the optical element support member 23 may be configured so that their operations are interlocked using a gear mechanism or a motor drive.
[0060]
The interlocking operation is preferably configured so that the optical path switching optical element 23b is inserted into the optical axis when the third relay lens system 24 is inserted into the optical axis.
[0061]
Note that this embodiment is merely an example, and is not limited to this configuration or shape, and can be appropriately modified and changed within the scope of the present invention.
[0062]
【The invention's effect】
As described above, according to the present invention, it is possible to provide a microscope that can be used for both a confocal laser scanning microscope and a total reflection microscope, and that can share a control device and the like, by simply switching optical members.
[Brief description of the drawings]
FIG. 1 is a schematic configuration diagram when a microscope according to a first embodiment of the present invention is used as a confocal laser scanning microscope.
FIG. 2 is a schematic configuration diagram when the microscope according to the first embodiment of the present invention is used as a total reflection microscope.
FIG. 3 is a schematic configuration diagram when a microscope according to a second embodiment of the present invention is used as a confocal laser scanning microscope.
FIG. 4 is a schematic configuration diagram when a microscope according to a second embodiment of the present invention is used as a total reflection microscope.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Laser illumination light source 2 Illumination light 3 Two-dimensional scanner 4 1st relay lens system 5 2nd relay lens system 6 Objective lens 7 Sample 8 Beam splitter 9 Imaging lens 10 Pinhole 11 Photomultiplier 12 Control circuit 13 Personal computer 14 Relay Lens system (third relay lens system)
15 Relay lens system (3rd relay lens system)
16 Cover glass 17 Beam splitter 18 Illumination light cut filter 19 Imaging lens 20 Imaging device 21 Image generation circuit 22 Lens support members 22a and 23a Rotating shaft 23b Optical path switching optical element 23 Optical element support member 24 Third relay lens system 25 Fixed relay Lens system 26 Lens support member

Claims (6)

A laser illumination light source;
An objective lens;
A scanner disposed between the laser illumination light source and the objective lens, and scanning the illumination light from the laser illumination light source in at least one direction;
A first relay lens system fixedly disposed between the scanner and the objective lens;
A second relay lens system disposed between the objective lens and the first relay lens system;
A third relay lens system arranged to be interchangeable with the second relay lens system;
The focal length of the second relay lens system is larger than the focal length of the first relay lens system,
When prior Symbol second relay lens system is inserted in the optical axis, the second relay lens system, the illumination light from the first relay lens system and the second relay lens system the objective lens Through the first relay lens system and the second relay lens system, the light deflection unit of the scanner and the entrance pupil plane of the objective lens. Is substantially optically conjugated, and the illumination light can scan and illuminate the specimen by the scanner,
When the third relay lens system is inserted into the optical axis, the third relay lens system transmits the illumination light from the first relay lens system to the objective lens via the third relay lens. The first relay lens system and the third relay lens system are arranged so as to collect light near the entrance pupil plane, and the optical deflection unit of the scanner and the focal plane on the sample side of the objective lens are approximately optically conjugate. The microscope is characterized in that the illumination light can totally illuminate the specimen. A laser illumination light source;
An objective lens;
A scanner disposed between the laser illumination light source and the objective lens, and scanning the illumination light from the laser illumination light source in at least one direction;
A first relay lens system fixedly disposed between the scanner and the objective lens;
A fixed relay lens system for condensing the illumination light from the first relay lens system on a focal plane on the specimen side of the objective lens ;
A third relay lens system for condensing the illumination light from the first relay lens system in the vicinity of an entrance pupil plane of the objective lens;
The third relay lens system is detachable between the first relay lens system and the fixed relay lens system,
When the third relay lens system is removed from the optical axis , the optical deflection unit of the scanner and the entrance pupil plane of the objective lens are substantially optically controlled by the first relay lens system and the fixed relay lens system. The illumination light can scan and illuminate the specimen by the scanner,
When the third relay lens system is inserted into the optical axis, the first relay lens system, the fixed relay lens system, and the third relay lens system allow the light deflection unit of the scanner and the objective lens to A microscope characterized in that a focal plane on the specimen side is substantially optically conjugate, and the illumination light can totally illuminate the specimen. A first observation optical system provided between the laser illumination light source and the scanner;
An observation optical path switching optical element detachably provided between the objective lens and the third relay lens system;
A second observation optical system provided in the vicinity of the observation optical path switching optical element,
When the second relay lens system or the fixed relay lens system is inserted in the optical axis and the observation optical path switching optical element is removed from the optical axis, light from the specimen is reflected in the first observation optical system. Can be observed
When the third relay lens system is inserted into the optical axis and the observation optical path switching optical element is inserted into the optical axis, light from the specimen passes through the observation optical path switching optical element. microscope according to claim 1 or 2, characterized in that can be observed in the observation optical system. The microscope according to claim 3 , wherein the observation optical path switching optical element includes a beam splitter and an illumination light cut filter arranged at approximately 45 degrees with respect to the optical axis. The said 3rd relay lens system is movable in an optical axis direction in order to condense the said illumination light to the entrance pupil plane vicinity of the said objective lens, The any one of the Claims 1 thru | or 4 characterized by the above-mentioned. Microscope. Only when the third relay lens system is inserted into the optical axis, the insertion / removal operation of the third relay lens system and the observation optical path switching element are inserted so that the observation optical path switching optical element is inserted into the optical axis. The microscope according to any one of claims 1 to 5 , wherein the insertion / removal operation is interlocked.

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